Europaisches Patentamt
3
European Patent Office
GO Publication number:
© Application number: 87107568.5
249
7 7 2
A 2
Office europeen des brevets
EUROPEAN
0
PATENT A P P L I C A T I O N
©
int. CI."C12N 5 / 0 0
@ Date of filing: 25.05.87
©
Priority: 09.06.86 US 871966
@ Date of publication of application:
23.12.87 Bulletin 87/52
©
Designated Contracting States:
BE CH DE ES FR GB IT LI NL SE
© Applicant: CPC INTERNATIONAL INC.
International Plaza P.O. Box 8000
Englewood Cliffs New Jersey 07632(US)
©
Inventor: Kinnersley, Alan M.
7702 W. 65th Place
Bedford Park, IL. 60499(US)
Inventor: Henderson, Wayne E.
674 Pontiac Lane
Bolingbrook, IL 60439(US)
©
Representative: Lederer, Franz, Dr. et al
Vanderwerth, Lederer & Riederer
Patentanwalte Lucile-Grahn-Strasse 22
D-8000 Munchen 80(DE)
© Method and composition for plant tissue and cell culture.
© Improved cell differentiation is achieved by culturing plant tissue and plant cells in a culture medium wherein
the carbohydrates comprise a mixture of maltose and glucose.
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0 249 772
METHOD AND COMPOSITION FOR PLANT TISSUE AND CELL CULTURE
The present invention relates to a method of plant tissue and cell culture which increases cell
differentiation and the amount of secondary products produced by the cells.
When plant cells are grown outside the plant fn synthetic media, undifferentiated ce!F multiplication,
called callus formation, is observed. The ceHs may also undergo plant cell differentiation with the
5 development in culture of embryos or one or more portions of the plant, generally roots or shoots.
Differentiation of plant tissue culture is of importance in agriculture where rapid plant propagation of
tissue culture is independent of seed availability and growing season. Plants produced from tissue culture
have uniform characteristics and are free of virus.
Another application of plant tissue culture which depends on cell differentiation is the production of high
value
chemicals. Accordingly, a method for enhancing plant cell differentiation in cell tissue culture may
ra
also be used as arr improved: method" for obtaining such chemical products.
The media an which plant tissue cultures are grown contain a carbohydrate component as the major
nutrient The carbohydrate uaia% comprises from 80 to 90% by weight of the solid constituents of the
media. The preferred carbohydrates for this purpose have been sucrose or glucose. Occasionally, workers
T5 have used maltose, lactose, or other simple sugars.
We have now discovered, surprisingly, that if a proper combination of maltose and glucose is used as
alt or part of the carbohydrate component of the medium, improved cell differentiation of the cell tissue
culture is observed. This results not only in enhanced production of roots and shoots, but also in the
increased production of secondary chemicals by the cells.
In accordance with this invention, there is provided an improved process for increasing cell differenti2a
ation in cell tissue cultures which comprises culturing a piece of tissue excised from a plant body, or cells
obtained by cultivation of tissue excised from a plant body, in a synthetic basal plant tissue culture medium
containing carbohydrates and other nutrients required for growth of said cell tissues wherein the carbohydrates comprise from about 20% to about 90% maltose by weight and at least about 10% glucose by
2s weight.
Further, in accordance with this invention, there is provided a medium for the growth of cell tissue
culture wherein the carbohydrates of said medium comprise from about 20% to about 90% maltose by
weight and at least about 10% glucose by weight.
According to the present invention, plant tissues and the cells are cultivated using a culture medium
30 wherein the carbohydrates in said medium comprise from about 20% to about 90% maltose by weight and
at least about t0% glucose by weight. The procedures for preparing the culture and culture medium may
be identical to those used to form conventional plant tissue cultures. Thus, a known synthetic culture
medium containing inorganic salts, micronutrients, vitamins, and hormones, can be used as the basic
culture medium, in which the carbohydrate is replaced with the carbohydrate mixture mentioned above.
The carbohydrate component used in the practice of this invention may be obtained in a variety of
35
ways. The desired amounts of dextrose and maltose can be combined to give a mixture with the desired
composition. Alternatively, certain starch hydrolyzates, which contain suitable combinations of dextrose and
maltose, can be employed. One such starch hydrolyzate which is commercially available from the Corn
Products Division of CPC International Inc. bears the trade name GLOBE® 1632. This hydrolyzate contains
40 approximately 35% dextrose and 30% maltose, with the remainder being higher oiigosaccharides. Carbohydrate mixtures suitable for use in this invention can also be obtained by mixing various amounts of dextrose
and maltose with starch hydrolyzates to give a mixture having the desired amounts of these sugars.
The amount of carbohydrate mixture containing maltose and dextrose to be added to the basal culture
medium, in accordance with the present invention, may be in the range of from about 10 grams to about 50
45 grams, preferably from about 15 grams to about 30 grams, per liter of medium. The culture medium to
which the carbohydrate has been added may be used per se for the cultivation. It is permissible, as in a
conventional culture, to incorporate further differentiation promoting agents, such as phytohormones, in the
medium in accordance with the specific purpose.
The plant tissue to be cultured includes any tissue taken out of an individual plant body, and particularly
so parts such as shoot apex, cambium, seedling hypocotyl, and the like are preferred. It is also possible to
cultivate undifferentiated cells developed in plants, such as callus cells. Tissues or cells resulting from
successive cultivation may be used as well.
0 249 772
When a plant tissue is cultivated in a culture medium containing the carbohydrate mixture of this
invention, the promotion of differentiation is realized, so that a plant body, stem, leaf, or root can be
developed from the callus. It is possible to harvest the cell mass and the grown body or parts, or to
.transplant the differentiated: plant body as a seedling. It may also be possible to extract useful substances
5" from the cells obtained.
As described above, the method according to the present invention permits an efficient cultivation of a
plant tissue or cells by enhancing their multiplication and differentiation by adding a proper carbohydrate
mixture to the culture medium. By this means, it is possible to harvest the cultivated plants or cells as such,
or to extract useful substances from them. It is also possible to obtain homogenous seedling cultures
w thereby propagating a particular plant species.
The following examples illustrate certain embodiments of the present invention. Unless otherwise stated,
all proportions and: percentages are provided on the basis of weight.
75
EXAMPLE t
Tobacco plants,. IMicotiana tabacum L. - cultivar Burley 21 , were grown from seed. Leaves were surfacesterilized by immersion in 0.3% NaOCI solution for 20 minutes followed by two washes with distilled water.
Explants were obtained with a 0.6-cm diameter cork borer and were cultured on a basic culture medium
20" solidified with 0.9% agar. The composition of the medium is given in Table I.
25
3JI1
35
40
45
50
55
0 249 772
TABLE r
BASrC CULTURE MEDIUM (pH 5 . 7 )
MiUfqrams/Liter
■
NH4NG3
TO
T5
20
KNO3
1,900
CaCT2*2H20
440
HqSQ/^'THiQ
370
KH2PQ4
170
Na2EDTA
37.3
FeSQ4-7tfz0
27.8
"
H3BO3
25
30
35
1,650
16.9
MnS04-H20
ZnSO4-7K2O
8.6
Kr
0.83
Na2MaO4.-2H2O
0.25
CuS04-5H20
0.025
CoCl2-6H20
0.025
Sucrose
40
50
55
30,000
i-Inositol
Nicotinic
45
6.2
100.0
Acid
0.5
Pyridoxine-HCl
0.5
Thfanrine-HCI
0.1
a 1 pha- Napht Hal e r t e a c e t i c Acid
2.0
Kinetin
0.2
The cells formed calli when grown on the solid media in test tubes in the dark at 25 °C for 2 months.
Suspension cultures were initiated from the calli by first dispersing the callus tissue in liquid medium using
a rolled tube on a rotating drum and then transferring a 2-5-ml aliquot of the suspension to a 250-ml
Erlenmeyer flask containing 50 ml of fresh medium. Flasks were kept in darkness at room temperature on a
gyrotary shaker at 100 rpm. Three to four flasks of each culture were maintained. The suspension cultures
were subcultured approximately every 20 days into fresh growth medium. During several months of serial
passage, nicotine was not detected in the tissue cultures.
(1 249 772
Nicotine analysis was performed by taking an 8-or 16-mi aliquot of a suspension culture, mixing it with
10 ml of 5 M NaOH, and steam distilling the- mixture. The steam distillate was collected in 5 ml of 0.5 M
HCI and the nicotine content of the distillate was determined spectrophotometrically by measuring the
ultraviolet absorption at 236, 259; and 2821 nrnr. Nicotine content was calculated from the absorbances by
standard, methods as reported by, Willits, etal, Anal, Chem., 22 , 430-433 (1950).
Cells which had grown under the- above- conditions for 8 months were transferred into growth media
which contained a starch hydrolyzate in place of the sucrose. This starch hydrolyzate contained 37%
glucose, 29% maltose, and 34% of higher oligosaccharides. Control cultures were run using sucrose as the
carbohydrate in the medium. Cultures containing both media were subcultured at approximately 20-day
TO intervals, and aliquots of the suspension cultures were analyzed for nicotine at each harvest date. The
results given in Table II, whictr are: the- average of three or four cultures in each case, show that only the
starch hydroiyzate containing, s. mixture of gJucose and maltose produced appreciable quantities of nicotine.
rs
TABLE I I
TOBACCO SUSPENSION CULTURES GROWN ON
SUCROSE AND STARCH HYDROLYZATE
20)
Tissue
25
Harvest
No.
Sucrose
in.
Mfedfum
1
2
3
4
5
6
7
8
9
12.1
11. I
6.1
15.1
"7.2
7.1
14.3
17.6
16.5
30
35
40
45
50
55
a)
Cultures
passage
(qr/T
"
Dry B a s i s )
Starch
Hydrolyzate
in
Medium
7.1
13.1
9.5
16.6
5.56
6.81
12.8
11.1
10.8
•
Nicotine
(% of Tissue Dry B a s i s )
Starch
Sucrose
Hydrolyzate
in
in
Medium
Medium
0.219
0
0
<0.05
<0.05
<0.05
0
0
<0.05
were exposed to e x c e s s i v e t e m p e r a t u r e
due to f a i l u r e of a g y r o t a r y s h a k e r .
-
0
0
0.255
0.843
0.17
3.2
0.53aJ
0.23
1.45
during
this
EXAMPLE 2
The general procedure of Example 1 was followed in which the carbohydrate was either pure glucose,
pure maltose, or mixtures of these. The cells grew poorly on maltose and produced no measurable amount
of nicotine. The cells grew well on glucose but produced only small amounts of nicotine. The yields of
nicotine by cells grown on glucose were similar to those produced by cells grown on sucrose given in
Example 1. The results given in Table III show that mixtures of glucose and maltose, and starch
hydrolyzates containing mixtures of glucose and maltose, within the ranges disclosed in this invention, can
be used to grow tobacco tissue cultures which produce surprisingly high levels of nicotine.
0 249 772
TABLE I I I
TOBACCO SUSPENSION CULTURES GROWN ON
VARIOUS CARBOHYDRATES
TO
Harvest
No.
61 ucose
1
2
3
4
5
6
—
0.001
0.059
0.074
0.038
0.15
T5
20
a)
25
15% Glucose
85% Maltose
—
—
1.34
1.37
4.24
5.04
Nicotine
(% of Tissue Dry B a s i s )
25% Glucose
50% Glucose
75% Maltose
50% Maltose
0.44
0.037
0.38
1.49
4.34
5.02
A s t a r c h hydro! y z a t e c o n t a i n i n g
34% h i g h e r o l i g o s a c c h a r i d e s .
0.22
0.42
1.41
2.59
4.26
4.68
37% g l u c o s e ,
Hydrolyzates
A«O
B°J
0.40
0.028
1.67
0.40
2.77
4.33
29% m a l t o s e ,
0.16
0.023
0.84
0.86
and
b) A s t a r c h h y d r o T y z a t e c o n t a i n i n g 25% g l u c o s e , 19% m a l t o s e , a n d
56% h i g h e r o l i g o s a c c h a r i d e s - c o m p a r a t i v e t e s t , not an e x a m p l e
of t h i s i n v e n t i o n .
m
EXAMPLE 3
35
40
45
50
55
Suspension cultures of wild carrot (Daucus carota L.) were grown using the same procedure as used
for the suspension cultures of tobacco cells in Example 1. The medium used was the wild carrot medium of
Wetherell, Plant Physiology, 44, 1734-1737 (1969) as given in Table IV. Again, the suspension cultures were
subcultured approximately every 20 days into fresh growth medium.
0 243 772
TABLE IV
WILD CftRRffT MEBWM (pH 5 . 8 )
MiTli g r a m s / L i t e r
10
75
4,000
KNO3
NH4C1
540
MgS04-7H20
185
CaCl2-2H2G
220
-
KH2P04
2ff
Na2EDTA
18.6
■
13.6
FeS04-7H20
25
30
35
MnS04-H2G
7.0
ZnS04-7H20
4.0
H3BO3
2.4
(NH4)6Mo7024-4H20
0.01
KI
0.38
CuS04-5H20
0.015
Thiamine-HCl
3.0
20,000
Carbohydrate
40
45
50
55
68
2,4-Dichlorophenoxyacetic
Acid
0.5
Anthocyanin pigment produced by the carrot cells was determined by the following analytical technique.
Five-ml aliquots of the suspension culture were diluted with 2 volumes of water before the cells were
collected by centrifugation. The cells were extracted with 10 ml of a solution containing 95% methanol, 4%
water, and 1% hydrochloric acid by volume. Extractions were performed at room temperature overnight.
These mixtures were centrifuged and the absorbances of the bright pink solutions were determined at 530
nm against a solvent blank. The results, using various carbohydrates in the medium, are given in Table V
where the relative pigment production of the various cell cultures are indicated by the absorbance per 100
ml of culture. They clearly show that cultures containing carbohydrates with the proportions of maltose and
dextrose specified in this invention cause differentiation of the wild carrot cells with enhanced and more
rapid production of pigment over those given by cells grown on media containing the single carbohydrates:
sucrose, glucose, or maltose.
0 249 772
TABLE V
MILD CARROT SUSPENSION CULTURES
GROWN ON VARIOUS CARBOHYDRATES
70
Anthocyanin Pigment
(A530/10p ml )
Harvest
Na.
Sucrose
Glucose
Maltose
52% Glucose
48% Maltose3)
Starch
Hydro! y z a t e b )
I
2.49
3.53
2.7
7.4
7.2
2
3.20
3.53
6.5
8.8
9.5
Used at
a level
IS
20
a)
25
b) Ccmtafns
of only
37% g l u c o s e ,
12 g/1
of medium.
29% m a l t o s e ,
and
34% h i g h e r
oligosaccharides.
EXAMPLES
30
35
Suspension cultures of an embryogenic wild carrot cell line were grown on the medium given in Table
IV except that the 2,4-dichiorophenoxyacetic acid was omitted. This allowed the cultures to form embryos.
After two subcultures on a medium containing a given carbohydrate, the embryos were separated from the
medium, washed with water, and dried. The results given in Table VI show that the starch hydrolyzate,
which contains proportions of maltose and dextrose within those specified in this invention, caused
increased embryo production over those given by cells grown on media containing the single carbohydrates: sucrose, glucose, or maltose.
TABLE VI
WILD CARROT SUSPENSION CULTURES
GROWN ON VARIOUS CARBOHYDRATES
40
45
Sucrose
Embryo
'
Glucose
222
182
Weight
(mq)a)
Maltose
Starch
Hydro l y z a t e b )
307
339
50
a)
Each value
cultures.
b)
C o n t a i n s 37% g l u c o s e ,
oligosaccharides.
55
is
the
average
weight
29% m a l t o s e ,
from t h r e e
and
separate
34% h i g h e r
0 249 772
Thus, it is apparent that there has been provided, in accordance with the invention, an improved
medium for plant tissue culture. While the invention has been described in conjunction with specific
embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to
those skilled in the art in light of the foregoing description. Accordingly, it is intended to include all such
alternatives, modifications, and' variations as set forth within the spirit and scope of the appended claims.
Claims
w
ts
20
25
30
35
40
45
50
55
1. A process for increasing cell differentiation in cell tissue cultures which comprises culturing a piece
of tissue excised from a plant body, or cells obtained by cultivation of tissue excised from a plant body, in a
synthetic basal plant tissue culture medium containing carbohydrates and other nutrients required for
growth of said cell tissues wherein the carbohydrates comprise from about 20% to about 90% maltose by
weight and at least about 10% glucose by weight.
2. A process according to claim 1 wherein the culture medium contains from about 5 grams to about 50
grams carbohydrate per liter of said culture medium.
3. A process according to claim t wherein the carbohydrate is a starch hydrolyzate.
4. The process of claim 1 wherein the cell tissue culture is a tobacco cell tissue culture.
5. The process of claim 1 wherein the tissue culture is a wild carrot tissue culture.
6. A medium for the growth of cell tissue cultures wherein the carbohydrate in said medium comprises
from about 20% to about 90% maltose by weight and at least about 10% glucose by weight.
7. A medium of claim 6 wherein the carbohydrate .comprises from about 5 grams to about 50 grams per
liter of said medium.
8. A medium of claim 6 wherein the carbohydrate comprises a starch hydrolyzate.